| 1. |
- Berglund, Anna-Karin, 1979, et al.
(author)
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Nucleotide pools dictate the identity and frequency of ribonucleotide incorporation in mitochondrial DNA. : Mapping ribonucleotides in mitochondrial DNA
- 2017
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In: PLoS genetics. - : Public Library of Science (PLoS). - 1553-7404 .- 1553-7390. ; 13:2
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Journal article (peer-reviewed)abstract
- Previous work has demonstrated the presence of ribonucleotides in human mitochondrial DNA (mtDNA) and in the present study we use a genome-wide approach to precisely map the location of these. We find that ribonucleotides are distributed evenly between the heavy- and light-strand of mtDNA. The relative levels of incorporated ribonucleotides reflect that DNA polymerase γ discriminates the four ribonucleotides differentially during DNA synthesis. The observed pattern is also dependent on the mitochondrial deoxyribonucleotide (dNTP) pools and disease-causing mutations that change these pools alter both the absolute and relative levels of incorporated ribonucleotides. Our analyses strongly suggest that DNA polymerase γ-dependent incorporation is the main source of ribonucleotides in mtDNA and argues against the existence of a mitochondrial ribonucleotide excision repair pathway in human cells. Furthermore, we clearly demonstrate that when dNTP pools are limiting, ribonucleotides serve as a source of building blocks to maintain DNA replication. Increased levels of embedded ribonucleotides in patient cells with disturbed nucleotide pools may contribute to a pathogenic mechanism that affects mtDNA stability and impair new rounds of mtDNA replication.
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| 2. |
- Chen, Song, et al.
(author)
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Compressive fatigue limit of four types of dental restorative materials
- 2016
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In: Journal of The Mechanical Behavior of Biomedical Materials. - : Elsevier BV. - 1751-6161 .- 1878-0180. ; 61, s. 283-289
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Journal article (peer-reviewed)abstract
- The purpose of this study was to evaluate the quasi-static compressive strength and the compressive fatigue limit of four different dental restorative materials, before and after aging in distilled water for 30 days. A conventional glass ionomer cement (Fuji IX GP; IG), a zinc-reinforced glass ionomer cement (Chemfil rock; CF), a light curable resin-reinforced glass ionomer cement (Fuji II LC; LC) and a resin-based composite (Quixfil; QF) were investigated. Cylindrical specimens (4 mm in diameter and 6 mm in height) were prepared according to the manufacturer's instructions. The compressive fatigue limit was obtained using the staircase method. Samples were tested in distilled water at 37 degrees C, at a frequency of 10 Hz with 10(5) cycles set as run-out. 17 fatigue samples were tested for each group. Two-way ANOVA and one-way ANOVA followed by Tukey's post-hoc test were used to analyze the results. Among the four types of materials, the resin-based composite exhibited the highest compressive strength (244 +/- 13.0 MPa) and compressive fatigue limit (134 +/- 7.8 MPa), followed by the light-cured resin reinforced glass ionomer cement (168 +/- 8.5 MPa and 92 +/- 6.6 MPa, respectively) after one day of storage in distilled water. After being stored for 30 days, all specimens showed an increase in compressive strength. Aging showed no effect on the compressive fatigue limit of the resin-based composite and the light-cured resin reinforced glass ionomer cement, however, the conventional glass ionomer cements showed a drastic decrease (37% for IG, 31% for CF) in compressive fatigue limit. In conclusion, in the present study, resin modified GIC and resin-based composite were found to have superior mechanical properties to conventional GIC.
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| 3. |
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| 4. |
- Engqvist, Martin, 1983, et al.
(author)
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Directed evolution of gloeobacter violaceus rhodopsin spectral properties
- 2015
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In: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 427:1, s. 205-220
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Journal article (peer-reviewed)abstract
- Proton-pumping rhodopsins (PPRs) are photoactive retinal-binding proteins that transport ions across biological membranes in response to light. These proteins are interesting for light-harvesting applications in bioenergy production, in optogenetics applications in neuroscience, and as fluorescent sensors of membrane potential. Little is known, however, about how the protein sequence determines the considerable variation in spectral properties of PPRs from different biological niches or how to engineer these properties in a given PPR. Here we report a comprehensive study of amino acid substitutions in the retinal-binding pocket of Gloeobacter violaceus rhodopsin (GR) that tune its spectral properties. Directed evolution generated 70 GR variants with absorption maxima shifted by up to ± 80 nm, extending the protein's light absorption significantly beyond the range of known natural PPRs. While proton-pumping activity was disrupted in many of the spectrally shifted variants, we identified single tuning mutations that incurred blue and red shifts of 42 nm and 22 nm, respectively, that did not disrupt proton pumping. Blue-shifting mutations were distributed evenly along the retinal molecule while red-shifting mutations were clustered near the residue K257, which forms a covalent bond with retinal through a Schiff base linkage. Thirty eight of the identified tuning mutations are not found in known microbial rhodopsins. We discovered a subset of red-shifted GRs that exhibit high levels of fluorescence relative to the WT (wild-type) protein.
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| 5. |
- Engqvist, Martin, 1983, et al.
(author)
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GLYCOLATE OXIDASE3, a Glycolate Oxidase Homolog of Yeast l-Lactate Cytochrome c Oxidoreductase, Supports l-Lactate Oxidation in Roots of Arabidopsis
- 2015
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In: Plant Physiology. - : Oxford University Press (OUP). - 1532-2548 .- 0032-0889. ; 169:2, s. 1042-1061
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Journal article (peer-reviewed)abstract
- In roots of Arabidopsis (Arabidopsis thaliana), L-lactate is generated by the reduction of pyruvate via L-lactate dehydrogenase, but this enzyme does not efficiently catalyze the reverse reaction. Here, we identify the Arabidopsis glycolate oxidase (GOX) paralogs GOX1, GOX2, and GOX3 as putative L-lactate-metabolizing enzymes based on their homology to CYB2, the L-lactate cytochrome c oxidoreductase from the yeast Saccharomyces cerevisiae. We found that GOX3 uses L-lactate with a similar efficiency to glycolate; in contrast, the photorespiratory isoforms GOX1 and GOX2, which share similar enzymatic properties, use glycolate with much higher efficiencies than L-lactate. The key factor making GOX3 more efficient with L-lactate than GOX1 and GOX2 is a 5- to 10-fold lower Km for the substrate. Consequently, only GOX3 can efficiently metabolize L-lactate at low intracellular concentrations. Isotope tracer experiments as well as substrate toxicity tests using GOX3 loss-offunction and overexpressor plants indicate that L-lactate is metabolized in vivo by GOX3. Moreover, GOX3 rescues the lethal growth phenotype of a yeast strain lacking CYB2, which cannot grow on L-lactate as a sole carbon source. GOX3 is predominantly present in roots and mature to aging leaves but is largely absent from young photosynthetic leaves, indicating that it plays a role predominantly in heterotrophic rather than autotrophic tissues, at least under standard growth conditions. In roots of plants grown under normoxic conditions, loss of function of GOX3 induces metabolic rearrangements that mirror wild-type responses under hypoxia. Thus, we identified GOX3 as the enzyme that metabolizes L-lactate to pyruvate in vivo and hypothesize that it may ensure the sustainment of low levels of L-lactate after its formation under normoxia.
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| 6. |
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| 7. |
- Kreisel, Katrin, 1991, et al.
(author)
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DNA polymerase η contributes to genome-wide lagging strand synthesis.
- 2019
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In: Nucleic acids research. - : Oxford University Press (OUP). - 1362-4962 .- 0305-1048. ; 47:5, s. 2425-2435
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Journal article (peer-reviewed)abstract
- DNA polymerase η (pol η) is best known for its ability to bypass UV-induced thymine-thymine (T-T) dimers and other bulky DNA lesions, but pol ηalso has other cellular roles. Here, we present evidence that pol η competes with DNA polymerases α and δfor the synthesis of the lagging strand genome-wide, where it also shows a preference for T-T in the DNA template. Moreover, we found that the C-terminus of pol η,which contains a PCNA-Interacting Protein motif is required for pol ηto function in lagging strand synthesis. Finally, we provide evidence that a pol η dependent signature is also found to be lagging strand specific in patients with skin cancer. Taken together, these findings provide insight into the physiological role of DNA synthesis by pol η and have implications for our understanding of how our genome is replicated to avoid mutagenesis, genome instability and cancer.
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| 8. |
- Kreisel, Katrin, 1991, et al.
(author)
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Simultaneous mapping and quantitation of ribonucleotides in human mitochondrial DNA
- 2017
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In: Journal of Visualized Experiments. - : MyJove Corporation. - 1940-087X. ; 2017
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Journal article (peer-reviewed)abstract
- © 2017. Established approaches to estimate the number of ribonucleotides present in a genome are limited to the quantitation of incorporated ribonucleotides using short synthetic DNA fragments or plasmids as templates and then extrapolating the results to the whole genome. Alternatively, the number of ribonucleotides present in a genome may be estimated using alkaline gels or Southern blots. More recent in vivo approaches employ Next-generation sequencing allowing genome-wide mapping of ribonucleotides, providing the position and identity of embedded ribonucleotides. However, they do not allow quantitation of the number of ribonucleotides which are incorporated into a genome. Here we describe how to simultaneously map and quantitate the number of ribonucleotides which are incorporated into human mitochondrial DNA in vivo by Next-generation sequencing. We use highly intact DNA and introduce sequence specific double strand breaks by digesting it with an endonuclease, subsequently hydrolyzing incorporated ribonucleotides with alkali. The generated ends are ligated with adapters and these ends are sequenced on a Next-generation sequencing machine. The absolute number of ribonucleotides can be calculated as the number of reads outside the recognition site per average number of reads at the recognition site for the sequence specific endonuclease. This protocol may also be utilized to map and quantitate free nicks in DNA and allows adaption to map other DNA lesions that can be processed to 5´-OH ends or 5´-phosphate ends. Furthermore, this method can be applied to any organism, given that a suitable reference genome is available. This protocol therefore provides an important tool to study DNA replication, 5´-end processing, DNA damage, and DNA repair.
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| 9. |
- Pires, Marcel V., et al.
(author)
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The influence of alternative pathways of respiration that utilize branched-chain amino acids following water shortage in Arabidopsis
- 2016
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In: Plant, Cell and Environment. - : Wiley. - 1365-3040 .- 0140-7791. ; 39:6, s. 1304-1319
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Journal article (peer-reviewed)abstract
- During dark-induced senescence isovaleryl-CoA dehydrogenase (IVDH) and D-2-hydroxyglutarate dehydrogenase (D-2HGDH) act as alternate electron donors to the ubiquinol pool via the electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF/ETFQO) pathway. However, the role of this pathway in response to other stresses still remains unclear. Here, we demonstrated that this alternative pathway is associated with tolerance to drought in Arabidopsis. In comparison with wild type (WT) and lines overexpressing D-2GHDH, loss-of-function etfqo-1, d2hgdh-2 and ivdh-1 mutants displayed compromised respiration rates and were more sensitive to drought. Our results demonstrated that an operational ETF/ETFQO pathway is associated with plants' ability to withstand drought and to recover growth once water becomes replete. Drought-induced metabolic reprogramming resulted in an increase in tricarboxylic acid (TCA) cycle intermediates and total amino acid levels, as well as decreases in protein, starch and nitrate contents. The enhanced levels of the branched-chain amino acids in loss-of-function mutants appear to be related to their increased utilization as substrates for the TCA cycle under water stress. Our results thus show that mitochondrial metabolism is highly active during drought stress responses and provide support for a role of alternative respiratory pathways within this response.
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| 10. |
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| 11. |
- Ting, Miriam, et al.
(author)
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Classification and Effects of Implant Surface Modification on the Bone : Human Cell-Based In Vitro Studies
- 2017
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In: Journal of Oral Implantology. - : ALLEN PRESS INC. - 0160-6972 .- 1548-1336. ; 43:1, s. 58-83
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Research review (peer-reviewed)abstract
- Implant surfaces are continuously being improved to achieve faster osseointegration and a stronger bone to implant interface. This review will present the various implant surfaces, the parameters for implant surface characterization, and the corresponding in vitro human cell-based studies determining the strength and quality of the bone-implant contact. These in vitro cell-based studies are the basis for animal and clinical studies and are the prelude to further reviews on how these surfaces would perform when subjected to the oral environment and functional loading.
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| 12. |
- Wanrooij, Paulina H., et al.
(author)
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Ribonucleotides incorporated by the yeast mitochondrial DNA polymerase are not repaired
- 2017
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In: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 114:47, s. 12466-12471
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Journal article (peer-reviewed)abstract
- Incorporation of ribonucleotides into DNA during genome replication is a significant source of genomic instability. The frequency of ribonucleotides in DNA is determined by deoxyribonucleoside triphosphate/ribonucleoside triphosphate (dNTP/rNTP) ratios, by the ability of DNA polymerases to discriminate against ribonucleotides, and by the capacity of repair mechanisms to remove incorporated ribonucleotides. To simultaneously compare how the nuclear and mitochondrial genomes incorporate and remove ribonucleotides, we challenged these processes by changing the balance of cellular dNTPs. Using a collection of yeast strains with altered dNTP pools, we discovered an inverse relationship between the concentration of individual dNTPs and the amount of the corresponding ribonucleotides incorporated in mitochondrial DNA, while in nuclear DNA the ribonucleotide pattern was only altered in the absence of ribonucleotide excision repair. Our analysis uncovers major differences in ribonucleotide repair between the two genomes and provides concrete evidence that yeast mitochondria lack mechanisms for removal of ribonucleotides incorporated by the mtDNA polymerase. Furthermore, as cytosolic dNTP pool imbalances were transmitted equally well into the nucleus and the mitochondria, our results support a view of the cytosolic and mitochondrial dNTP pools in frequent exchange. © 2017, National Academy of Sciences. All rights reserved.
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